1. Field of the Invention
The present invention relates to a molecular detection chip including a metal oxide semiconductor field effect transistor (MOSFET) formed on sidewalls of a micro-fluid channel, and a molecular detection device employing the molecular detection chip. Also, the present invention relates to a molecular detection method using the molecular detection device, and more particularly, to a quantitative detection method for the immobilization of molecular probes or the binding of molecular probes and a target sample. The present invention relates to a nucleic acid mutation assay device constructed by incorporating a thermal control and detection unit including a heater and a thermal sensor into the molecular detection device, and a nucleic acid mutation assay method using the nucleic acid mutation assay device.
2. Description of the Related Art
The disclosure of the human DNA sequence by the completion of genome project has accelerated researches into the function of diverse nucleic acids and proteins coded by the nucleic acids, and at the same time, increased the need for biosensors for easy detection of biomolecules such as nucleic acids and proteins.
Biosensors capable of sensing biomolecules using electrical signals are disclosed in U.S. Pat. Nos. 4,238,757, 4,777,019, 5,431,883, and 5,827,482. U.S. Pat. No. 4,238,757 discloses a field effect transistor (FET) designed to have a source and drain, including a layer of antibodies specific to a particular antigen. The concentration of antigens in a sample solution is measured from drain current variations over time using the FET.
U.S. Pat. No. 4,777,019 discloses a FET in which a gate is formed across doped source and drain regions, and a nucleotide complementary to a target nucleotide to be measured is bound to the top of the gate.
U.S. Pat. No. 5,431,883 discloses a FET in which a thin film of phthalocyanin, an organic insulating material capable of being changed to be conductive through reactions with chemical species, is formed to connect a gate and a drain.
U.S. Pat. No. 5,827,482 discloses a biosensor including two FETs connected in parallel, each having respective gates to which molecular receptors sensitive to different materials are bound for improved sensitivities.
However, currently available biosensors are all formed as conventional planar surface FETs each having a source, a drain, and a channel layer on the surface of a planar substrate so that high integration of the biosensors is restricted. In addition, it is difficult to selectively immobilize biomolecular probes on a limited region. Accordingly, immobilization of probes on the FETs is performed by using a separate fabrication apparatus in the fabrication of the FETs. However, the resulting probes are weakly immobilized on the FETs so that binding to a target molecule cannot be detected with high sensitivity. In addition, a considerable time is required to check for the probe immobilization, thereby increasing the overall time consumption for target molecule detection.
Therefore, there is an increasing need for a new biosensor which is easy to highly integrate, ensures stable immobilization of probes and in-situ confirmation of the probe immobilization, and can detect binding of a target molecule with high sensitivity.
Biochips refer to chips having highly immobilized biomolecular probes, such as DNA, proteins, etc., to be analyzed on substrates and are used for the analysis of a gene expression profile, genetic defects, a protein profile, and reaction patterns. Biochips can be categorized into a microarray chip having immobilized probes on a solid substrate and a lab-on-a-chip having immobilized probes on a micro-channel according to the type of immobilization of the probes, and into a DNA chip, a protein chip, etc., according to the kinds of probes.
Most DNA chips currently available are manufactured based on a spotting or photolithography technique. A DNA chip is manufactured by immobilizing only a single DNA strand that can react with a target DNA, as a probe on a particular substrate using chemical bonds, and detects the target DNA from the reaction. In manufacturing such a DNA chip, immobilization of probe DNAs greatly affects the reliability and reproducibility of products, and thus it needs to be accurately controlled. However, techniques in current use fail to accurately quantify the immobilized biomolecules.
Conventional spotting chips or photolithography chips can adjust the quantity of probes to a certain level on a volume basis in the manufacturing process, but have poor accuracy and reproducibility for use as commercial products for the diagnosis of diseases. In particular, for the investigation of particular DNA expression, more accurate immobilization of probe DNAs is required. Despite the need for an accurate immobilization technique, one has not been established yet due to technical problems in manufacturing processes and cost concerns.
To overcome the conventional problems, the present inventors have conducted research and completed the present invention where the voltage and current characteristics of a DNA chip were measured using a MOSFET sensor in the DNA chip so that immobilization and hybridization of probe DNAs could be accurately detected. As a result, a DNA chip capable of measuring the immobilization and hybridization of probe DNAs at the same time can be manufactured for commercial uses without an increase in the manufacturing cost.
Single nucleotide polymorphism (SNP) of nucleic acids, which is a single base pair variation of human DNA between individuals, is the most common DNA sequence polymorphism (about 1 per 1000 bases). SNP affects the immune system of individuals and thus can be effective for diagnosing, treating, and preventing inherited diseases. Therefore, there is a need for a rapid and convenient detection method of SNP originating from individual or racial genetic differences and immunities.
Common SNP detection methods are based on temperature-dependent separation of double-stranded DNA. Double-stranded DNA is separated into two single strands at a temperature greater than about 95° C. Based upon these characteristics, the sequence of mutated DNA can be identified. However, this method needs discrete systems and apparatuses for each step and cannot be applied for real-time DNA separation and immediate detection through accurate and precise temperature control.
U.S. Pat. No. 6,171,850 entitled “Integrated Devices and Systems for performing Temperature Controlled Reactions and Analyses” discloses use of a heat exchanger in controlling the internal and external temperatures of individual reactors. The reaction system includes a heater and at least one heat exchanger. This reaction system is merely focused on temperature control of a plurality of reactors. Also, the inclusion of the heat exchanger is not advantageous in DNA detection and improvement of fluid channel characteristics.
U.S. Pat. No. 6,174,676 entitled “Electrical Current for Controlling Fluid Parameters in Micro-channels” discloses a variety of heaters and a fluid channel equipped with an electrically controlled heater and cooler. This patent is restricted to the temperature control apparatus for PCR without description of a device for detecting temperature-based separation of DNA duplex.
U.S. Pat. No. 5,683,657 entitled “DNA meltometer” discloses a nucleic acid analytical device including a temperature control chamber for carrying a buffer solution while being kept at a predetermined temperature, a heating and cooling unit for controlling the temperature of the temperature control chamber, and a labeling and detecting unit for labeling thermally denaturated double-stranded DNA at a temperature Tm with fluorescent materials and detecting them. In the DNA meltometer, the DNA detecting unit is separated from the temperature control chamber, thereby complicating the overall system and causing a delay in the detection step.
To address these problems, the present inventors have developed a DNA mutation (SNP) assay device which ensures DNA detection as well as temperature adjustment in a variety of ways based upon the fact that a double-stranded DNA is separated into two single strands at an increased temperature. According to the present invention, whether DNA is separated or not can be identified in real time using a DNA detection unit disposed on a fluid channel.